Bioengineering & Translational Medicine最新文献

{"title":"Fecal microbiota transplantation for the treatment of intestinal and extra‐intestinal diseases: Mechanism basis, clinical application, and potential prospect","authors":"Dongxin Yi, Tao Li, Yuji Xiao, Xue Zhang, Qiangqiang Hao, Feng Zhang, Tianming Qiu, Guang Yang, Xiance Sun, Ying Dong, Ningning Wang","doi":"10.1002/btm2.10728","DOIUrl":"https://doi.org/10.1002/btm2.10728","url":null,"abstract":"To review the theoretical basis and therapeutic effects of fecal microbiota transplantation (FMT) in various diseases in animal experiments and clinical studies, as well as the limitations and current standards of FMT application. PubMed and Web of Science databases were searched for articles published only in English between 1975 and 2023 on reliable results of animal experiments and clinical treatment of FMT. The properties of the gut microbiota and its interactions with the host metabolism are critical to human health, and microbiome disturbance is closely associated with human intestinal and extra‐intestinal diseases. Therefore, therapeutic tools targeting on the modulation of gut microbiota have attracted increasing attention, among which FMT represents the most widely studied intervention strategy. This review gathered and summarized application of FMT in intestinal diseases, metabolic diseases, hypertension, cancer, nervous system diseases and arthritis, and elaborated the beneficial effects that can be achieved by altering the microbiota with FMT and the mechanisms of action. In addition, the potential risks and side effects of FMT approach are discussed, as well as current efforts to standardize the development of FMT. Through a systemic review of the outcome and mechanism of FMT in the treatment of intestinal diseases and extra‐intestinal diseases, we aimed to provide a theoretical basis for the construction of an optimized FMT framework, so as to better exert its application prospects.","PeriodicalId":9263,"journal":{"name":"Bioengineering & Translational Medicine","volume":"70 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142597117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ColMA‐based bioprinted 3D scaffold allowed to study tenogenic events in human tendon stem cells","authors":"Giacomo Cortella, Erwin Pavel Lamparelli, Maria Camilla Ciardulli, Joseph Lovecchio, Emanuele Giordano, Nicola Maffulli, Giovanna Della Porta","doi":"10.1002/btm2.10723","DOIUrl":"https://doi.org/10.1002/btm2.10723","url":null,"abstract":"The advent of bioprinting has enabled the creation of precise three‐dimensional (3D) cell cultures suitable for biomimetic in vitro models. In this study, we developed a novel protocol for 3D printing methacrylated collagen (ColMa, or PhotoCol®) combined with tendon stem/progenitor cells (hTSPCs) derived from human tendon explants. Although pure ColMa has not previously been proposed as a printable hydrogel, this paper outlines a robust and highly reproducible pipeline for bioprinting this material. Indeed, we successfully fabricated a 3D bioengineered scaffold and cultured it for 21 days under perfusion conditions with medium supplemented with growth/differentiation factor‐5 (GDF‐5). This bioprinting pipeline and the culture conditions created an exceptionally favorable 3D environment, enabling the cells to proliferate, exhibit tenogenic behaviors, and produce a new collagen type I matrix, thereby remodeling the surrounding environment. Indeed, over the 21‐day culture period under perfusion condition, tenomodulin expression showed a significant upregulation on day 7, with a 2.3‐fold increase, compared to days 14 and 21. Collagen type I gene expression was upregulated nearly 10‐fold by day 14. This trend was further confirmed by western blot analysis, which revealed a statistically significant difference in tenomodulin expression between day 21 and both day 7 and day 14. For type I collagen, significant differences were observed between day 0 and day 21, as well as between day 0 and day 14, with a <jats:italic>p</jats:italic>‐value of 0.01. These results indicate a progressive over‐expression of type I collagen, reflecting cell differentiation towards a proper tenogenic phenotype. Cytokines, such as IL‐8 and IL‐6, levels peaked at 8566 and 7636 pg/mL, respectively, on day 7, before decreasing to 54 and 46 pg/mL by day 21. Overall, the data suggest that the novel ColMa bioprinting protocol effectively provided a conducive environment for the growth and proper differentiation of hTSPCs, showcasing its potential for studying cell behavior and tenogenic differentiation.","PeriodicalId":9263,"journal":{"name":"Bioengineering & Translational Medicine","volume":"42 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142561830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Facile minocycline deployment in gingiva using a dissolvable microneedle patch for the adjunctive treatment of periodontal disease","authors":"Huimin Li, Xueyu Wen, Xinyi Gong, Yange Wu, Puxuan Zhao, Yun Zhang, Zhuomin Sha, Hao Chang, Xuepeng Chen","doi":"10.1002/btm2.10730","DOIUrl":"https://doi.org/10.1002/btm2.10730","url":null,"abstract":"Minocycline is a commonly used drug for adjunctive therapy in periodontal disease. However, the current mainstream local medications primarily rely on intra‐pocket administration, which, while avoiding the side effects of traditional systemic drugs, presents challenges such as inconvenience, discomfort, and the need for professional assistance, thus affecting patient compliance. Herein, we introduce a minocycline‐loaded dissolvable microneedle (Mino‐DMN) patch that allows for local and efficient delivery of minocycline to gingiva for the treatment of periodontitis. A two‐step casting micro‐molding process involving vacuum drying and freeze drying is employed to concentrate minocycline in the microneedle part and limit its diffusion into the patch backing. The resulting Mino‐DMN patch features an array of minocycline‐enriched gelatin MNs with a porous HA patch backing. The microneedles can penetrate into gingiva with enough mechanical strength and quickly release minocycline into the gingival tissue, ensuring prolonged local residence of the drug and minimizing its loss to saliva. In vivo experiments show Mino‐DMN inhibits pro‐inflammatory factors, promotes anti‐inflammatory factors, and stimulates bone formation, surpassing topical application and comparable to the inconvenient and discomfort administration of Periocline®. This proposed Mino‐DMN offers a simple, efficient, user‐friendly strategy for the adjunctive treatment of periodontal disease.","PeriodicalId":9263,"journal":{"name":"Bioengineering & Translational Medicine","volume":"33 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142486726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Temperature‐sensitive sodium beta‐glycerophosphate/chitosan hydrogel loaded with all‐trans retinoic acid regulates Pin1 to inhibit the formation of spinal cord injury‐induced rat glial scar","authors":"Rongmou Zhang, Ting Tang, Huafeng Zhuang, Peiwen Wang, Haiming Yu, Hao Xu, Xuedong Yao","doi":"10.1002/btm2.10729","DOIUrl":"https://doi.org/10.1002/btm2.10729","url":null,"abstract":"Glial scar formation is a major obstacle to nerve regeneration following spinal cord injury (SCI). Pin1 and the PI3K/AKT/CDK2 signaling pathway play crucial roles in neuronal regulation, but research on their involvement in glial scarring remains limited. In this study, we have for the first time observed that Pin1, PI3K, AKT, and CDK2 are upregulated and interact with each other following SCI. Further experiments revealed that Pin1 contributes to the development of glial scars by promoting astrocyte proliferation, inhibiting apoptosis, and activating the PI3K/AKT/CDK2 pathway. Additionally, all‐trans retinoic acid (ATRA), a specific chemical inhibitor of Pin1, effectively suppresses Pin1 expression. However, its clinical application is limited by its short half‐life and susceptibility to inactivation. To address these issues, we have developed a thermosensitive sodium beta‐glycerophosphate (β‐GP)/chitosan (CS) hydrogel loaded with ATRA (β‐GP/CS@ATRA). This hydrogel exhibits favorable morphology and biocompatibility. Compared to free ATRA, the β‐GP/CS@ATRA hydrogel significantly enhances functional motor recovery after SCI and protects spinal cord tissue, thereby inhibiting glial scar formation. Mechanistically, ATRA administration blocks the development of glial scars and the activation of the PI3K/AKT/CDK2 pathway by inhibiting Pin1 expression. This study suggests that combining ATRA with a hydrogel to target Pin1 expression may be a promising strategy for treating glial scar formation following SCI.","PeriodicalId":9263,"journal":{"name":"Bioengineering & Translational Medicine","volume":"23 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142448565","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Recent regulatory developments in EU Medical Device Regulation and their impact on biomaterials translation","authors":"Klaudia M. Jurczak, Torben A. B. van der Boon, Raul Devia‐Rodriguez, Richte C. L. Schuurmann, Jelmer Sjollema, Lidia van Huizen, Jean‐Paul P. M. De Vries, Patrick van Rijn","doi":"10.1002/btm2.10721","DOIUrl":"https://doi.org/10.1002/btm2.10721","url":null,"abstract":"We envision this work to assist researchers and medical device developers (beside other stakeholders) to better understand biomaterial‐based medical device development and its approval process proposed by the new MDR and IVDR in the European Union, as more complex biomaterials emerge, with the MDR reflecting the progress in biomaterial discoveries. Additionally, insufficient international harmonization in regulatory laws and poor‐quality data reporting contribute to the problem. This review describes the possible reasons for a slowing biomaterials translational trend observed over the past decades, focusing on the European Market, and suggests a feasible approach for biomaterials‐based medical device translation into the clinic. Suitable solutions to upgrade biomaterial translation to the clinic have not yet been provided by the field: no additional hurdles should be imposed for researchers, clinicians, the medical device industry, and insurance companies, which all should collaborate on bringing innovative solutions to patients. The new MDR and IVDR represent a substantial advancement in ensuring patient safety and reflect a major step forward in healthcare. However, they should not constrain innovation in biomaterials‐based medical device development. Incorporating reverse engineering from patient safety and a ‘safe by design’ (SbD) strategy early into medical device development might lead to a smoother and successful approval process. A solid R&amp;D phase, with an emphasis on device safety and performance assessment, is fundamental to ensure an effective transition into the clinic. We offer an overview of the recently implemented regulations on medical devices and in vitro diagnostics across the EU, describing a shifting paradigm in the field of biomaterials discovery. As more complex biomaterials emerge, suitable regulations will be necessary to keep bringing safe and well‐performing medical solutions to patients.","PeriodicalId":9263,"journal":{"name":"Bioengineering & Translational Medicine","volume":"18 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142443860","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Photobiomodulation improves functional recovery after mild traumatic brain injury","authors":"Andrew R. Stevens, Mohammed Hadis, Abhinav Thareja, Freya G. Anderson, Michael R. Milward, Valentina Di Pietro, Antonio Belli, William Palin, David J. Davies, Zubair Ahmed","doi":"10.1002/btm2.10727","DOIUrl":"https://doi.org/10.1002/btm2.10727","url":null,"abstract":"Mild traumatic brain injury (mTBI) is a common consequence of head injury but there are no recognized interventions to promote recovery of the brain. We previously showed that photobiomodulation (PBM) significantly reduced the number of apoptotic cells in adult rat hippocampal organotypic slice cultures. In this study, we first optimized PBM delivery parameters for use in mTBI, conducting cadaveric studies to calibrate 660 and 810 nm lasers for transcutaneous delivery of PBM to the cortical surface. We then used an in vivo weight drop mTBI model in adult rats and delivered daily optimized doses of 660, 810 nm, or combined 660/810 nm PBM. Functional recovery was assessed using novel object recognition (NOR) and beam balance tests, whilst histology and immunohistochemistry were used to assess the mTBI neuropathology. We found that PBM at 810, 660 nm, or 810/660 nm all significantly improved both NOR and beam balance performance, with 810 nm PBM having the greatest effects. Histology demonstrated no overt structural damage in the brain after mTBI, however, immunohistochemistry using brain sections showed significantly reduced activation of both CD11b<jats:sup>+</jats:sup> microglia and glial fibrillary acidic protein (GFAP)<jats:sup>+</jats:sup> astrocytes at 3 days post‐injury. Significantly reduced cortical localization of the apoptosis marker, cleaved caspase‐3, and modest reductions in extracellular matrix deposition after PBM treatment, limited to choroid plexus and periventricular areas were also observed. Our results demonstrate that 810 nm PBM optimally improved functional outcomes after mTBI, reduced markers associated with apoptosis and astrocyte/microglial activation, and thus may be useful as a potential regenerative therapy.","PeriodicalId":9263,"journal":{"name":"Bioengineering & Translational Medicine","volume":"9 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142415629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The hanging‐heart chip: A portable microfluidic device for high‐throughput generation of contractile embryonic stem cell‐derived cardiac spheroids","authors":"Pei‐Tzu Lai, Cheng‐Kun He, Chi‐Han Li, Jefunnie Matahum, Chia‐Yu Tang, Chia‐Hsien Hsu","doi":"10.1002/btm2.10726","DOIUrl":"https://doi.org/10.1002/btm2.10726","url":null,"abstract":"Stem cell‐derived cardiac spheroids are promising models for cardiac research and drug testing. However, generating contracting cardiac spheroids remains challenging because of the laborious experimental procedure. Here, we present a microfluidic hanging‐heart chip (HH‐chip) that uses a microchannel and flow‐driven system to facilitate cell loading and culture medium replacement operations to reduce the laborious manual handling involved in the generation of a large quantity of cardiac spheroids. The effectiveness of the HH‐chip was demonstrated by simultaneously forming 50 mouse embryonic stem cell‐derived embryonic bodies, which sequentially differentiated into 90% beating cardiac spheroids within 15 days of culture on the chip. A comparison of our HH‐chip method with traditional hanging‐drop and low‐attachment plate methods revealed that the HH‐chip could generate higher contracting proportions of cardiac spheroids with higher expression of cardiac markers. Additionally, we verified that the contraction frequencies of the cardiac spheroids generated from the HH‐chip were sensitive to cardiotoxic drugs. Overall, our results suggest that the microfluidic hanging drop chip‐based approach is a high‐throughput and highly efficient method for generating contracting mouse embryonic stem cell‐derived cardiac spheroids for cardiac toxicity and drug testing applications.","PeriodicalId":9263,"journal":{"name":"Bioengineering & Translational Medicine","volume":"17 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142385478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cardiovascular patches applied in congenital cardiac surgery: Current materials and prospects","authors":"Mingze Sun, Vincent Reed LaSala, Caroline Giuglaris, David Blitzer, Sophia Jackman, Senay Ustunel, Kavya Rajesh, David Kalfa","doi":"10.1002/btm2.10706","DOIUrl":"https://doi.org/10.1002/btm2.10706","url":null,"abstract":"Congenital Heart Defects (CHDs) are the most common congenital anomalies, affecting between 4 and 75 per 1000 live births. Cardiovascular patches (CVPs) are frequently used as part of the surgical armamentarium to reconstruct cardiovascular structures to correct CHDs in pediatric patients. This review aims to evaluate the history of cardiovascular patches, currently available options, clinical applications, and important features of these patches. Performance and outcomes of different patch materials are assessed to provide reference points for clinicians. The target audience includes clinicians seeking data on clinical performance as they make choices between different patch products, as well as scientists and engineers working to develop patches or synthesize new patch materials.","PeriodicalId":9263,"journal":{"name":"Bioengineering & Translational Medicine","volume":"26 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142360302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A CRISPR mediated point‐of‐care assay for the detection of mucosal calprotectin in an animal model of ulcerative colitis","authors":"Selena Chia, Tianruo Guo, Ewa M. Goldys, Sophie C. Payne, Wenlu Duan, Nigel H. Lovell, Mohit N. Shivdasani, Fei Deng","doi":"10.1002/btm2.10725","DOIUrl":"https://doi.org/10.1002/btm2.10725","url":null,"abstract":"Inflammatory bowel disease (IBD) is a chronic disorder associated with inflammation in the gastrointestinal tract, leading to a range of debilitating symptoms. Fecal calprotectin is an established biomarker for ulcerative colitis (UC), one of the main IBD diseases, which provides indications of the presence and severity of inflammation in the digestive tract. Enzyme‐Linked Immunosorbent Assay (ELISA) as a gold standard approach for fecal calprotectin detection is time‐consuming and impractical in point‐of‐care settings. Moreover, obtaining fecal samples from patients is challenging and inhibits longitudinal monitoring. To address these specific problems, we have developed a novel approach for detecting calprotectin which leverages clustered regularly interspaced short palindromic repeats (CRISPR)/Cas technology. We successfully developed a portable tube‐based CRISPR/Cas assay for point‐of‐care testing of calprotectin. This assay showed a detection range from 1 to 10,000 ng/ml (over 4 log units), using both fluorescent and colorimetric analytical techniques. The established assay was further validated through measurements in mucosal samples obtained in an anesthetised preclinical rodent model of UC, with 2–3 times higher calprotectin concentration detected in UC rat samples compared to that of healthy control animals. This point‐of‐care test may provide a rapid, precise, and user‐friendly approach for the diagnosis and monitoring of IBD through mucosal sample testing.","PeriodicalId":9263,"journal":{"name":"Bioengineering & Translational Medicine","volume":"3 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142321313","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A novel cytoprotective organ perfusion platform for reconstructing homeostasis of DCD liver while alleviating IRI injury","authors":"Tingting Lan, Mingxing Yu, Tao Ming, Hong Wang, Juan Deng, Shuhan Cheng, Zhongyang Shen, Deling Kong","doi":"10.1002/btm2.10724","DOIUrl":"https://doi.org/10.1002/btm2.10724","url":null,"abstract":"Pump is a vital component for expelling the perfusate in small animal isolated organ normothermic machine perfusion (NMP) systems whose flexible structure and rhythmic contraction play a crucial role in maintaining perfusion system homeostasis. However, the continuous extrusion forming with the rigid stationary shaft of the peristaltic pumps can damage cells, leading to metabolic disorders and eventual dysfunction of transplanted organs. Here, we developed a novel biomimetic blood‐gas system (BBGs) for preventing cell damage. This system mimics the cardiac cycle and features an adjustable inspiratory‐to‐expiratory (IE) ratio to mitigate acidosis caused by continuous oxygen inhalation. In our study, adipose stem cells (ADSCs) were cultured within the circulatory system for 10 min, 2, and 4 h. Compared to the peristaltic pump, the BBGs significantly reduced cell apoptosis and morphological injury while enhancing cell proliferation and adhesion. Additionally, when the supernatant from ADSCs was introduced to LPS‐induced macrophages for 24 h, the BBGs group demonstrated a more pronounced anti‐inflammatory effect, characterized by reduced M1 macrophage expression. Besides, with isolated rat livers from donation after circulatory death (DCD) perfusion with ADSCs for 6 h by the BBGs, we detected fewer apoptotic cells and a reduced inflammatory response, evidenced by down‐regulated TNF‐α expression. The development of BBGs demonstrates the feasibility of recreating physiological liquid–gas circulation in vitro, offering an alternative platform for isolated organ perfusion, especially for applications involving cell therapy.","PeriodicalId":9263,"journal":{"name":"Bioengineering & Translational Medicine","volume":"9 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142276853","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}